Gerald L. Lamoureux

Propachlor metabolism in soybean plants, excised soybean tissues, and soil

Abstract Propachlor was rapidly metabolized to a homoglutathione conjugate in the roots and foliage of soybean plants. No other competing reactions were observed. The homoglutathione conjugate was rapidly metabolized to the cysteine conjugate which was slowly converted to a variety of other metabolites, four of which were present up to 72 days after treatment. Those four metabolites were the malonylcysteine, malonylcysteine S -oxide, 3-sulfinyllactic acid, and the O -malonylglucoside conjugates of propachlor. A cysteine S -oxide conjugate was also observed as a transient metabolite. Fast atom bombardment mass spectrometry was used to characterize the polar metabolites. Some metabolites were isolated and identified from both soybean plants and peanut cell suspension cultures. The terminal metabolites in soybean plants were concentrated in the roots and foliage. Less than 1% of the metabolites were present in the beans and pods of the mature plants. In a sandy loam soil, propachlor and the cysteine conjugate of propachlor were both metabolized in approximately the same ratio to the bound residue fraction and three major metabolites: N -isopropyloxanilic acid, 2-sulfo- N -isopropylacetanilide, and 2-(sulfinylmethylenecarboxy)- N -isopropylacetanilide. The methyl sulfoxide and methyl sulfone analogs of propachlor were characterized as minor soil metabolites. The major metabolites present in soybean plants grown in soil treated with propachlor were those produced in the soil and taken up by the plants. It was speculated that propachlor metabolism in the soil involved microbial activity and conjugation with glutathione or cysteine. It was concluded that homoglutathione conjugates are metabolized in soybean in a manner similar to the metabolism of other glutathione conjugates in species such as peanut.

Mercapturic acid formation in the metabolism of propachlor, CDAA, and fluorodifen in the rat

Abstract When [ 14 C]F 3 -fluorodifen (2,4′-dinitro-4-trifluoromethyl diphenylether), carbonyl-[ 14 C]CDAA ( N,N -diallyl-2-chloroacetamide), and carbonyl- 14 C-propachlor (2-chloro- N -isopropylacetanilide) were fed to rats, 57 to 86% of the 14 C was excreted via the urine within 48 hr. Although very little radioactivity was excreted in the feces of CDAA-treated rats, 15–22% of the 14 C was excreted in the feces of propachlor- of fluorodifentreated rats and an average of 8% of the 14 C remained in these rats 48 hr after treatment. Oxidation of the 14 C label to [ 14 C]O 2 was not a major process in the metabolism of these herbicides. The only major radioactive metabolite present in the 24-h urine of fluorodifen-treated rats, 2-nitro-4-trifluoromethylphenyl mercapturic acid, accounted for 41% of the administered dose of 14 C. In the metabolism of CDAA, the corresponding mercapturic acid accounted for 76% of the dose; it was the only major metabolite present in the 24-h urine. In contrast, three major metabolites were detected in the 24-h urine of propachlortreated rats, and the mercapturic acid accounted for only 20% of the dose. The mercapturic acid of each herbicide was identified by mass spectrometry.

GLUTATHIONE AND GLUCOSIDE CONJUGATION IN HERBICIDE SELECTIVITY

The conjugation of herbicides with glutathione or glucose are frequently species specific reactions that result in herbicide detoxification. Therefore, glutathione and glucoside conjugation play an important role in herbicide detoxification and selectivity. Phase 1 activation reactions are sometimes necessary before glutathione or glucoside conjugation can occur. In these cases, the Phase 1 reaction rather than conjugation may be responsible for herbicide selectivity. This appears to be particularly true in the metabolism of herbicides to 0-glucoside conjugates. The glutathione-S-transferases and glucosyltransferases that catalyze these conjugation reactions, the role that these reactions play in the selectivity of specific herbicides, factors that affect these reactions, and the secondary metabolism of glutathione and glucoside conjugates are reviewed.

Diphenyl ether herbicide metabolism in a spruce cell suspension culture: The identification of two novel metabolites derived from a glutathione conjugate☆

Abstract [ CF 3 - 14 C]Fluorodifen herbicide (2,4′-dinitro-4-trifluoromethyldiphenyl ether) was rapidly metabolized by a spruce cell suspension culture ( Picea abies L. Karst). The primary route of metabolism involved cleavage of the diphenyl ether bond by glutathione (GSH). The resulting conjugate of fluorodifen ( S -[4-trifluoromethyl-2-nitrophenyl]glutathione) appeared to be metabolized sequentially to the corresponding γ-glutamylcysteine conjugate, the cysteine conjugate, and two novel metabolites. These novel metabolites, identified by fast atom bombardment mass spectrometry and enzyme hydrolysis, were the S -glucoside and the S -(3-thio-2- O -glucosyl) lactic acid conjugate of 4-trifluoromethyl-2-nitrobenzene. This appears to be the first report on the plant metabolism of GSH conjugates to metabolites of these classes. During the metabolism of [ 14 C]fluorodifen, a loss of water-soluble 14 C-labeled metabolites from the spruce cells to the growth medium was observed. After 1 day, 63% of the applied 14 C was present as metabolites in the cells and 30% was present as metabolites in the medium. However, after 10 days only 14% of the applied 14 C was present as metabolites in the cells and 50% was present as metabolites in the medium. During this period, the recovery of 14 C declined from 97% after 1 day to 64% after 10 days. The decrease in recovery may have been due to the loss of volatile metabolites such as 4-trifluoromethyl-2-nitrothiophenol. The major metabolites present after 1 day were the GSH conjugate (36%), the γ-glutamylcysteine conjugate (35%), and the cysteine conjugate (14%). After 10 days, the major metabolites were the S -glucoside (45%) and the S -(2- O -glucosyl)-3-thiolactic acid conjugate (12%).

Synergism of diazinon toxicity and inhibition of diazinon metabolism in the house fly by tridiphane: Inhibition of glutathione S-transferase activity☆

Abstract Diazinon toxicity to a susceptible strain of house fly ( Musca domestica L.) was synergized by tridiphane [2-(3,5-dichlorophenyl)-2-(2,2,2-trichloroethyl)oxirane], a herbicide synergist. Both diazinon and tridiphane were partially metabolized in the house fly by glutathione (GSH) conjugation. Synergism appeared to be due to inhibition of diazinon metabolism/detoxification. Crude glutathione S -transferase (GST) preparations from the house fly catalyzed GSH conjugation of diazinon, tridiphane, 3,4-dichloronitrobenzene (DCNB), and chloro-2,4-dinitrobenzene (CDNB). Tridiphane and the GSH conjugate of tridiphane appeared to inhibit diazinon GSH conjugation, but diazinon did not inhibit tridiphane GSH conjugation. The enzymatic rate of tridiphane GSH conjugation was 22 times the rate of diazinon GSH conjugation; therefore, attempts to assay tridiphane as an inhibitor of diazinon GSH conjugation were inconclusive because of the high concentration of tridiphane GSH conjugate produced during the assay. CDNB underwent enzymatic GSH conjugation at a rate 240 times faster than that of tridiphane and 5000 times faster than that of diazinon. GSH conjugation of CDNB was not inhibited by tridiphane, but was inhibited by the GSH conjugate of tridiphane. In vivo , the GSH conjugate of tridiphane was produced in sufficient concentration to cause the observed inhibition of diazinon metabolism and synergism of diazinon toxicity. However, the possibility that parent tridiphane caused or contributed to the inhibition of diazinon metabolism and synergism of diazinon toxicity could not be excluded. Inhibition of diazinon metabolism did not appear to be due to depletion of either GSH or GST.

The effect of CDAA (N,N,-diallyl-2-chloroacetamide) pretreatments on subsequent CDAA injury to corn (Zea mays L.)

Abstract The effects of CDAA ( N,N -diallyl-2-chloroacetamide) pretreatment on subsequent CDAA injury to corn were examined and compared with the effects of the herbicide protectant R-25788 ( N,N ,-diallyl-2,2-dichloroacetamide). In addition, the effects of CDAA pretreatment on subsequent CDAA metabolism were determined. It was found that 5μ M CDAA protected corn from injury by 200 μ M CDAA when given as a 2.5- or 1-day pretreatment. R-25788 at similar concentrations did not protect corn from subsequent R-25788 injury. Pretreatment with CDAA increased GSH levels of corn roots by 61% within 1 day, and these levels did not increase with a longer 2.5-day pretreatment with CDAA. GSH- S -transferase activity was assayed spectrophotometrically using CDNB (1-chloro-2,4-dinitrobenzene). A 1-day pretreatment with CDAA increased the root GSH- S -transferase activity by 35%, but did not affect shoot GSH- S -transferase activity. A 2.5-day pretreatment resulted in a 50% increase in root GSH- S -transferase activity but no response of the shoot enzyme was observed. Even longer pretreatments with CDAA did not result in any further increases in enzyme activity. When corn roots pretreated with CDAA for 2.5 days were excised and incubated with radiolabeled CDAA, they exhibited greater rates of uptake and metabolism than did nonpretreated roots. With in vitro studies, a fairly high rate of nonenzymatic degradation of CDAA was observed. However, the enzymatic rate was always double that of the nonenzymatic rate under the experimental conditions used. It is concluded that elevations in the GSH levels and GSH- S -transferase activities of corn roots following CDAA pretreatments may be involved in the protection of corn from subsequent CDAA injury.

Metabolism of substituted diphenylether herbicides in plants. II. Identification of a new fluorodifen metabolite, S-(2-nitro-4-trifluoromethylphenyl)-glutathione in peanut

Abstract The herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), is eleaved in peanut to give the metabolite, S -(2-nitro-4-trifluoromethylphenyl)-glutathione. A comparison of the glutathione conjugate isolated from treated peanut leaves and from in vitro pea epicotyl glutathione S -transferase reaction showed that both metabolites were identical. Other polar metabolites were also isolated, but not identified. The structure of the glutathione conjugate was confirmed by amino acid analysis and by mass, NMR, and infrared spectroscopy. The p -nitrophenyl moiety is also conjugated to natural products and is released as the free p -nitrophenol upon acid hydrolysis.

Glutathione S-transferase activity in spruce needles

Abstract Glutathione S -transferase activity was present in extracts from needles of two different spruce species ( Picea abies and Picea glauca ). In vitro conjugation studies were conducted with three 14 C herbicides and one 14 C fungicide: atrazine (2-chloro-4-ethylamino-6-isopropylamino- s -triazine), fluorodifen (2,4′-dinitro-4-trifluoromethyl diphenyl-ether), propachlor (2-chloro- N -isopropylacetanilide), and pentachloronitrobenzene (PCNB). The enzymes from both P. abies and P. glauca showed the highest rates of enzymatic conjugation for fluorodifen as the substrate while intermediate to low rates of enzymatic conjugation were observed with PCNB and propachlor. Atrazine was not an appreciable substrate for the enzymes of either species. The water-soluble 14 C conjugation products of the enzymatic reactions were assayed by liquid scintillation spectrometry. The [ 14 C]glutathione conjugates from fluorodifen and PCNB were identified by a combination of thinlayer chromatography (TLC), high-performance liquid chromatography (HPLC), and fast atom bombardment mass spectrometry and the [ 14 C]glutathione conjugate of propachlor was identified by TLC and HPLC comparison to an authentic standard. The catalytic properties of glutathione S -transferase from P. abies were analyzed with CDNB as substrate. The apparent K M values were 0.14 m M for GSH and 0.67 m M for CDNB, respectively, the pH optimum was between 7.6 and 8.0, and the temperature optimum was 40–45°C. The activation energy was calculated to be 32.4 kJ mol −1 .

In vitro metabolism of pentachloronitrobenzene to pentachloromethylthiobenzene by onion: Characterization of glutathione S-transferase, cysteine C-S lyase, and S-adenosylmethionine methyl transferase activities☆

Abstract Pentachloromethylthiobenzene (PCTA) was synthesized in vitro from pentachloronitrobenzene (PCNB) at pH 7.9 by an enzyme system from onion root that required dithiothreitol, glutathione, and S -adenosylmethionine. The soluble enzyme system was isolated from onion root by ammonium sulfate fractionation and differential centrifugation. The system contained glutathione S -transferase activity with PCNB, C-S lyase activity with S -(pentachlorophenyl)cysteine, S -adenosylmethionine methyl transferase activity with pentachlorothiophenol (PCTP), and presumably several peptidase activities. All activities were stable when the crude enzyme system was stored at −25°C. Evidence for the following sequence of reactions in PCTA synthesis was presented: PCNB→ 1 S -(pentachlorophenyl)glutathione→ 2 S -(pentachlorophenyl)-γ-glutamylcysteine→ 3 S -(pentachlorophenyl)cysteine→ 4 PCTP→ 5 PCTA. The first reaction was studied with [ 14 C]PCNB. Reactions 2–4 were studied with S -([ 14 C]pentachlorophenyl)glutathione, S -([ 14 C]pentachlorophenyl)cysteine, and peptide inhibitors. Reaction 5 was studied with [ 14 C]PCTP, S -[ 14 C]adenosylmethionine, and inhibitors. The possible use of the enzyme system in the characterization of other glutathione conjugates was discussed.

Mode of action of the dichloroacetamide antidote BAS 145-138 in corn: I. Growth responses and fate of metazachlor

The effect of BAS 145-138 (BAS) on metazachlor injury to corn and on the fate of [14C]metazachlor in corn seedlings was investigated. Corn shoot and root growth were inhibited by metazachlor. The antidote, BAS, increased corn shoot and root tolerance to metazachlor 10.7- and 7.6-fold, respectively. The antidotal activities of BAS and dichlormid were similar. Corn seedlings grown in soil treated with [14C]metazachlor ± BAS were dissected at two growth stages prior to emergence and one growth stage immediately after emergence. Parent [14C]metazachlor was present as <6% of the total radioactivity with an estimated tissue concentration of <1 μM in all tissues except the pericarp. This suggests that metazachlor was metabolized rapidly in both antidoted and control plants and that a very low concentration of metazachlor is required for phytotoxicity. BAS treatment reduced the concentration of parent metazachlor in the developing leaves by 82–84%. BAS treatment had three effects that contributed to the reduced amount of parent [14C]metazachlor in the developing leaves: (i) shoot absorption of [14C]metazachlor was slightly reduced by antidote treatment; (ii) the mobility of 14C was reduced in antidoted seedlings, as indicated by the 63–86% decrease of total 14C reaching the developing leaves; (iii) metabolism of metazachlor in growing tissues may have been stimulated by BAS, as suggested by the lower percentage of 14C present as parent metazachlor. The coleoptile plays a critical role in corn shoot tolerance to metazachlor, since more metazachlor is absorbed through the coleoptile than through the mesocotyl and corn is more sensitive to metazachlor absorbed through the coleoptile than the mesocotyl. Reduced absorption and movement of metazachlor through the coleoptile apparently contribute to antidote activity. Results are consistent with the hypothesis that BAS protects corn from metazachlor injury by reducing levels of parent metazachlor present in sensitive and rapidly growing tissues such as the developing leaves.